Abstract
Nitric oxide (NO) is a gaseous signaling molecule in plants, transducing information as a result of exposure to low temperatures. However, the underlying molecular mechanism linking NO with chilling stress is not well understood. Here, we functionally characterized the cucumber (Cucumis sativus L.) nitric oxide synthase-associated gene, NITRIC OXIDE ASSOCIATED 1 (CsNOA1). Expression analysis of CsNOA1, using quantitative real-time PCR, in situ hybridization, and a promoter::β-glucuronidase (GUS) reporter assay, revealed that it is expressed mainly in the root and shoot apical meristem (SAM), and that expression is up-regulated by low temperatures. A CsNOA1-GFP fusion protein was found to be localized in the mitochondria, and ectopic expression of CsNOA1 in the A. thaliana noa1 mutant partially rescued the normal phenotype. When overexpressing CsNOA1 in the Atnoa1 mutant under normal condition, no obvious phenotypic differences was observed between its wild type and transgenic plants. However, the leaves from mutant plant grown under chilling conditions showed hydrophanous spots and wilting. Physiology tolerance markers, chlorophyll fluorescence parameter (Fv/Fm), and electrolyte leakage, were observed to dramatically change, compared mutant to overexpressing lines. Transgenic cucumber plants revealed that the gene is required by seedlings to tolerate chilling stress: constitutive over-expression of CsNOA1 led to a greater accumulation of soluble sugars, starch, and an up-regulation of Cold-regulatory C-repeat binding factor3 (CBF3) expression as well as a lower chilling damage index (CI). Conversely, suppression of CsNOA1 expression resulted in the opposite phenotype and a reduced NO content compared to wild type plants. Those results suggest that CsNOA1 regulates cucumber seedlings chilling tolerance. Additionally, under normal condition, we took several classic inhibitors to perform, and detect endogenous NO levels in wild type cucumber seedling. The results suggest that generation of endogenous NO in cucumber leaves occurs largely independently in the (CsNOA1) and nitrate reductase (NR) pathway.
Highlights
Plants are frequently exposed to adverse environmental conditions that can limit growth and development, among which low temperature is a key factor
An alignment of the CsNOA1 amino acid sequence with apparent nitric oxide associated1 (NOA1) homologs from other plant species (Supplementary Figure S1B) showed that CsNOA1, AtNOA1, and OsNOA1 share three domains that are typical of the GTPase family: the zinc-binding domain (ZBD), the circularly permuted G-domain (CPG), and the C-terminal domain (CTD) (Moreau et al, 2008; Sudhamsu et al, 2008; Anand et al, 2009) (Supplementary Figure S1B)
Arabidopsis thaliana NO synthase1 (AtNOS1) was considered as plant NO synthase (NOS) (Guo et al, 2003) and is yet to be proven
Summary
Plants are frequently exposed to adverse environmental conditions that can limit growth and development, among which low temperature is a key factor. Plants can set up physiological and biochemical adaptations to cope with the stress challenge (Ruelland et al, 2009; Theocharis et al, 2012). These adaptations include changes in membrane composition, the induction of anti-oxidative systems, and the synthesis of protective molecules. It has been reported that transcriptional regulation of the carbohydrate metabolic pathway Arabidopsis thaliana is essential for the accumulation of specific carbohydrates that represent an important factor in improved tolerance to chilling stress (Cook et al, 2004; Maruyama et al, 2009, 2014). There is interest in identifying and characterizing the signaling network underlying the low temperature stress and there is growing evidence that nitric oxide (NO) is an important signal for transducing information related to low temperature exposure
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